Oral modified SSP-5 antibacterial composition

ABSTRACT

The present invention provides a composition for an oral cavity that contains the amino acid sequence of SEQ ID NO: 1, which is safe and effective without imparting bacterial resistance.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a composition for an oral cavity, whichsuppresses initial adhesion of intraoral bacteria to teeth andperiodontal tissue. The present invention further proves a method ofpreventing formation of a biofilm in an oral cavity.

2. Description of the Conventional Art

A biofilm is a film covering the surface of a microbial cell inconjunction with a substance or a precipitate secreted by bacteria whenmicroorganisms (i.e., bacteria or the like) adhere to the surface of anobject or an organism tissue and proliferate. An example of a typicalbiofilm in an oral cavity is plaque (bacterial plaque).

The genesis of oral cavity biofilim formation of the biofilm is anorganic ingredient, such as protein or the like, in saliva coming intocontact with the enamel surface, followed by a part of the organicingredient adhering to the enamel, and thereby forming a pellicle. Whenan intraoral bacteria (e.g., Streptococcus sanguis) in the saliva comesinto proximity with a tooth and contacts the pellicle, the intraoralbacteria is adsorbed to the pellicle, and a part of the bacteria adhereson the pellicle as it is. The adhered intraoral bacteria grows to thestrong biofilm adhering to other bacteria. The adhesion of the otherbacteria typically results from polysaccharides having tackyadhesiveness. These polysaccharides are knows as glucan or fructan andare made by using the nutrient or the like in the saliva. Subsequently,the bacteria in the biofilm repeats the proliferation to produce acid.Thereby, dental caries, periodontal disease or the like results.

Various method shave been researched to solve these problems. Forexample, an antibacterial agent and its assistant agent were proposed.As the assistant agent, antibacterial and antifungal assistants wereproposed as a composition for an oral cavity including a biofilmsuppressing assistant such as xylitol, farnesol or the like (forexample, refer to Japanese Patent Laid Open No. 2002-302404,2002-284604). However, since these antibacterial and antifungalassistants primarily serve to remove the biofilm, use of theantibacterial agent is indispensable.

It is known that the antibacterial agent problematically generatesbacterial resistance and also sterilizes indigenous bacteria existing inthe oral cavity. Therefore, there are limits to the use of antibacterialagents in a composition for an oral cavity.

Additional studies have attempted to add a protease, which is an enzymedecomposing protein, to an oral composition (for example, refer toJapanese Patent Laid Open No. 06-262165). However, since a protease isan enzyme, and as such there are problems associated with the timerequired to produce an effective result. Further, since proteases do notgenerally discriminate on the basis of protein identity beneficialproteins contained in the saliva are also decomposed.

Another attempt at solving the aforementioned problems have centered onthe use of monoclonal antibodies (for example, refer to Japanese PatentLaid Open No. 2002-114709). The monoclonal antibody suppressesglucosyltransferase, which is enzyme secreted by Streptococcus mutansbacteria when producing glucan. However, if the biofilm is alreadyformed, the effect with respect to the proliferation of the intraoralbacterial is hardly obtained. Additionally, the use of monoclonalantibodies also raises safety concerns since the monoclonal antibodyoriginating in the mouse is applied to the inside of an oral cavity ofperson.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide acomposition for an oral cavity that does not impart resistance to theobjective bacteria, is safe for a human body, and is effective within ashort time. Further, it is also an objective that this compositioneffectively suppress the formation of the biofilm, but does not reactwith indigenous bacteria and saliva that are not related to theformation of the biofilm in an oral cavity.

The earnest work was carried out and, as the result, a peptidecomprising a specific amino acid sequence that is capable of suppressingthe formation of the biofilm was found, by taking notice of thefollowing point. That is, when the intraoral bacteria forming thebiofilm is not sterilized but a substance suppressing an initialadhesion of bacteria to the oral cavity tissue is used, the formation ofthe biofilm can be suppressed. Then, the investigation was completed.

More particularly, it is known that Streptococcus bacteria in theintraoral bacteria has a strong initial adhesion to the oral cavitytissue, for example a tooth surface covered with the pellicle. Thebiofilm is formed by adhesion of these bacteria to the pellicle on thetooth surface giving opportunities for further adhesion of otherbacteria. Therefore, it is important to suppress the initial adhesion ofthe Streptococcus bacteria to the oral cavity tissue. For example, itwas confirmed that the initial adhesion of the Streptococcus mutansdepends on protein existing on the surface of bacteria, which is calledas PAc (Protein Antigen Serotype C) with the molecular weight of about190,000.

The present inventors researched the initial adhesion based on the Aarea of the PAc. On the basis of this research, it was determined that aspecific peptide part (hereinafter, referred to as SSP-5(390–402)) inthe protein is directly related to the initial adhesion of theStreptococcus intraoral bacteria to the tooth surface. ThisSSP-5(390–402) represents one fragment of the protein on the surface ofthe Streptococcus sanguis bacteria and has the sequence shown in SEQ IDNO: 1, wherein the residue corresponding to position 400 in SSP-5(residue 11 in SEQ ID NO: 1) is a threonine. The present inventors havemodified the original SSP-5 by isolated from Streptococcus sanguisbacteria for the further discussion herein (hereinafter, referred to asSEQ ID NO: 1 or modified SSP-5(390–402)).

As a result, when this peptide comprising the specific amino acidsequence is previously applied to the inside of an oral cavity, such asthe tooth surface or the like, this peptide can block the tooth surfacefrom Streptococcus intraoral bacteria adhering thereon, because theStreptococcus intraoral bacteria usually adhere on the tooth surfaceusing this peptide fragment but cannot adhere since the tooth surfacehas already been covered by this peptide fragment. Thus it is possibleto effectively suppress the initial adhesion of bacteria in a shorttime.

Therefore, one object of the present invention is to provide acomposition for an oral cavity, which contains a peptide comprising SEQID NO: 1.

The composition for an oral cavity according to the present inventionprovides a safety advantage when administered to a host organism ascompared with the conventional antibacterial agent since the compositionof the present invention is based on amino acids. When the compositionaccording to the present invention is applied to the tooth surface orthe like, the composition can effectively suppress the initial adhesionof bacteria in a short time, because the peptide covers the toothsurface for blocking before the bacteria are placed in contact with thetooth surface. Further, the composition according to the presentinvention is an excellent composition for an oral cavity capable ofeffectively suppressing the formation of the biofilm without reacting toindigenous bacteria and saliva at all. These indigenous bacteria andsaliva do not relate to the formation of the biofilm in an oral cavity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The method of producing the peptide comprising the specific amino acidsequence (modified SSP-5(390–402)) used in the composition for an oralcavity according to the present invention it is not limited. However, anamino acid synthesizer is preferably used. At the time of synthesizingthe peptide, it is important that the peptide does not contain anexcessive amino acid sequence beyond that of SEQ ID NO: 1. If there is apart of the excessive amino acid sequence, the original effect forsuppressing the formation of the biofilm cannot be obtained. Of course,a nonspecific peptide with respect to the initial adhesion of bacteria,which does not prevent the effect of the present invention, may beconnected to one end or both ends of SEQ ID NO: 1.

The composition for an oral cavity according to the present inventioncan be used in various forms based on existing methodologies. Thecomposition may be used by only blending a peptide of SEQ ID NO: 1 withwater or a solvent that is safe for a human body. This composition foran oral cavity may also be used as a tooth powder, a toothpaste, aliquid dentifrice or the like. The other blending ingredients are notlimited, so long as the blending ingredients do not prevent the functionof the peptide. The ingredients conventionally used in the compositionfor an oral cavity can be blended freely. More particularly, whenpreparing a toothpaste, abrasives, caking additives, wetting agents,sweeteners, perfumes, antiseptics, other pharmaceutically effectiveagents or the like can be suitably used.

More particularly, when preparing a tooth powder, the peptide of SEQ IDNO: 1 may be blended to the abrasives, such as calcium carbonate,calcium silicate, a silica fine powder, amorphous water-containingsilica, hydrophobic silica, calcium secondary phosphate, calciumpyrophosphate, insoluble sodium metaphosphate, aluminum hydroxide,silicic acid anhydride, or the like.

When preparing a toothpaste, the peptide of SEQ ID NO: 1 may be blendedusing the following as a base material in general. Base materials forthis purpose include water, glycerin, ethylene glycol, diethyleneglycol, polyethylene glycol, propylene glycol, polypropylene glycol,sorbitol, xylite, lactite, mannitol, ethanol, sodium lactate or thelike. To the base material, the abrasives used to the tooth powder maybe blended, if necessary.

Further, various kinds of a surfactant, such as an anionic surfactant, anonionic surfactant, an amphoteric surfactant, a semipolar surfactant orthe like, and a thickener may be suitably blended.

Examples of the anionic surfactant are an α-sulfofatty acid alkyl esteror its water-soluble salt, an alkyl sulfate ester salt, an N-acylaminoacid salt, or the like.

Examples of the nonionic surfactant are a fatty acid monoglyceride, afatty acid alkanol amide, a polyoxyethylene sorbitan fatty acid ester, apolyoxyethylene hardened castor oil, a saccharide fatty acid ester, orthe like.

As the amphoteric surfactant, an alkylbetanine, imidazolinium betaine, asulfobetaine, or the like are used.

As the semipolar surfactant, an alkylamine oxide or the like is used.

Examples of the thickener are sodium alginate, propylene glycol alginateester, carboxymethyl cellulose, carboxymethylcellulose sodium, carboxymethylcellulose calcium, starch sodium glycolate, starch phosphate estersodium, sodium polyacrylate, carboxypolymethylene, methylcellulose,crystalline cellulose, hydroxypropylcellulose, polyvinyl pyrrolidone,guar gum, carob bean gum, Tara gum, tamarind seed gum, gum Arabic,tragacanth gum, karaya gum, alginic acid, carrageenan, xanthan gum,gellan gum, curdlan, lactose, chitin, chitosan, chitosamine, or thelike.

Further, a germicide, an antiinflammatory agent, a phosphoric acidcompound and inorganic salts may be blended as pharmaceuticallyeffective ingredients.

Examples of the germicide are sodium monofluorophosphate, sodiumfluoride, chlorhexidine salts, benzethonium chloride, cetylpyridiniumchloride, benzalkonium chloride, or the like.

As the antiinflammatory agent, allantoinate or the like is used.

Examples of the phosphoric acid compound are sodium phosphate, sodiumpyrophosphate, sodium tripolyphosphate, sodium tetrapolyphosphate, orthe like.

As the inorganic salts, sodium chloride or the like is used.

Of course, a coloring matter, a presevative or the like may be added tothe composition for an oral cavity according to the present invention.

The composition for an oral cavity according to the present inventioncan be used as dental cleaning agents or medicines other than theabove-mentioned kinds of tooth powder and toothpaste. These dentalcleaning agents or medicines include a mouthwash, a chewing gum, agargle, a troche, a cream, an ointment, a pasting agent and a tablet.

As for the peptide comprising the specific amino sequence (SEQ ID NO: 1)in the composition for an oral cavity according to the presentinvention, the blending amount is 0.001–20% by weight preferably, andmore preferably 0.01–5% by weight.

Where the composition is a medicine, the amount of the peptide when usedfor internal use ranges from 5–50 mg per adult per day, and the amountof the peptide when used for external use is 1 to several 10 mg a time.

The peptide of the present invention can be used for preventing thedental caries or periodontal disease without giving harmful sideeffects. In the case of the tooth powder, the toothpaste or the like,the peptide can be used according to the ordinary method, consideringthese usage amounts.

EXAMPLE

Hereinafter, the composition for an oral cavity according to the presentinvention is explained with examples more concretely. However, thepresent invention is not limited to the following examples.

Example 1

<<Synthesis of the Peptide>>

A peptide comprising an amino acid sequence of SEQ ID NO: 1 was obtainedby a stepwise solid-phase peptide synthetic method. The synthesis wasconducted with a reversed phase HPLC (0.1% TFA of gradient of 10–45%acetonitrile) with a TSK-GEL column (30×1), by using Model 350 MultiplePeptide Synthesizer (Advanced Chemitech, Louisville, Ky., USA) as asynthesizer. The final purification degree was determined to be 95% ormore, by using reversed phase HPLC.

<<Confirmation of Adhesion Suppressing Properties>>

<Preparation of Bacteria>

S. sanguis (ATCC10556, ATCC10558), S. mutans (ATCC25175) were used asintraoral bacteria. All bacteria were cultured under an anaerobicconditions using Brain Heart Infusion broth (BHI; Difco Lavoratory,Detroit, Mich.) as a culture medium.

<Preparation of Saliva>

The specimen was stimulus saliva secreted by biting paraffine wax for 3minutes by 3 subjects (A, B, C). The saliva was collected, incubated at4° C. for 5 minutes, and centrifuged at 10000×g for 10 minutes at 4° C.Then, the supernatant was used the saliva specimen.

<Sensor Chip>

The adhesion suppressing properties of bacteria was confirmed by using asensor chip as a substitute for a tooth. As the sensor chip, PioneerSensor Chip F1 (produced by BIAcore Company) was used. The sensor chipF1 was activated at flow speed of 10 μL for 1 minute with 70 μL of 400mM N-ethyl-N′-(3-diaethylaminoprophyl) carbodiimide and 100 mM N-hydroxysiccinimide solution. Then, the above saliva specimen was diluted 20times, and bonded on the chip by passing 70 μL of sample over theactivated chip. (Hereinafter, this chip was said to as a salivatreatment sensor chip)

<Confirmation Method of the Adhesion Suppressing Properties>

The confirmation method of the adhesion suppressing properties by BIAcore™ Biosensor system (produced by BIAcore Company) will be explained.First, each bacteria is passed over the saliva treatment sensor chip at0.D.=1 (550 nm) and a flow speed of 20 μL/min. Then, the bonding state(Response Unit:RU) between the bacteria and saliva on the surface of thesensor chip was measured. The sensor chip was not carried out thepeptide treatment.

Then, to the other saliva treatment sensor chip, which was differentfrom the saliva treatment sensor chip adhered with bacteria, a modifiedSSP-5(390–402) peptide solution (1 mg/mL) dissolved with a phosphatebuffer solution (PBS, pH7.4) was passed over the sensor chip at flowspeed of 10 μL/min, and the surface of the saliva treatment sensor chipwas treated with the peptide. Then, each bacteria was passed over thechip at 0.D.=1 (550 nm) and flow speed of 20 μL/min, and the bondingstate (Response Unit:RU) between each bacteria and saliva on the sensorsurface was measured. The final bacterium adhesion suppressing ratio bythe peptide treatment was measured using the following formula 1.Adhesion suppressing ratio=100×{[Bonding state between bacteria andsaliva (RU)]−[Bonding state between bacteria and saliva (after thepeptide treatment) (RU)]}/[Bonding state between bacteria and saliva(RU)]  [Formula 1]

TABLE 1 Adhesion suppressing ratio Saliva S. sanguis S. sanguis S.mutans sample (ATCC10556) (ATCC10558) (ATCC25175) A 61% 59% 55% B 57%63% 60% C 65% 61% 58%

Example 2

As the composition for an oral cavity containing the peptide of SEQ IDNO: 1, the toothpaste was prepared with following compositions. Theadhesion suppressing properties of bacterial plaque was evaluated by theexperiment described below.

Calcium carbonate   40% by weight Sodium carboxymethyl cellulose  1.0%by weight Glycerin  8.0% by weight Sodium lauryl sulfate  1.5% by weightSorbitol 10.0% by weight Menthol  0.3% by weight Peptide comprising aspecific amino sequence  1.0% by weight Water 38.2% by weight

Example 3

As the composition for an oral cavity containing the peptide of SEQ IDNO: 1, a tablet-shaped composition for an oral cavity was prepared withfollowing compositions. The adhesion suppressing properties of bacterialplaque was evaluated by the experiment described below.

Lactose 65% by weight Crystalline cellulose 26% by weight Magnesiumstearate (lubricant)  4% by weight Xylitol  3% by weight Peptidecomprising a specific amino sequence  2% by weight

The effect of the composition for an oral cavity of Example 3 wasconfirmed by the following method.

The method comprising, washing and polishing of the teeth surfaces of 3subjects (A, B, C) in the dental clinic, brushing of only the left sideteeth in an oral cavity of each 3 subjects after 3 time meals per day byusing the toothpaste of Example 2, brushing of the right side teethusing a toothpaste, in which water was blended instead of the peptidecomprising the specific amino sequence of Example 2, measuring ofbacterial plaques of left and right side teeth with eyes by using adyeing method with a dye for an oral cavity (PROSPEC GEL, produced by GCCorporation) after one weak, and comparing of these bacterial plaques.These results were shown in Table 2 collectively.

TABLE 2 Left side in an oral Right side in an oral Subjects cavitycavity A Bacterial plaque is Bacterial plaque is hardly dyed colored reda little and can be confirmed B Bacterial plaque is Bacterial plaque ishardly dyed colored red a little and can be confirmed C Bacterial plaqueis Bacterial plaque is hardly dyed colored red a little and can beconfirmed

The effect of the composition for an oral cavity of Example 3 wasconfirmed by the following method.

The method comprising, washing and polishing of the teeth surfaces of 3subjects (D, E, F) in the dental clinic, brushing of the teeth of each 3subjects 3 times per day without using the toothpaste, and confirming ofthe amounts of bacterial plaques by the dyeing method after one weaklike the above-mentioned method. Then, the evaluation was continued bythe method comprising, washing and polishing of the teeth surfaces of 3subjects in the dental clinic again, brushing of the teeth after 3 timemeals per day without using the toothpaste, eating the tablet-shapedcomposition for an oral cavity of Example 3 3 times per day, andconfirming of the amounts of bacterial plaques by the dyeing method withthe dye for an oral cavity (PROSPEC GEL, produced by GC Corpotation)after one weak. These results were shown in Table 3 collectively.

TABLE 3 Composition for an oral Composition for an oral Subjects cavitywas not used cavity was used D Bacterial plaque is Bacterial plaque ishardly colored red a little and dyed can be confirmed E Bacterial plaqueis Bacterial plaque is hardly colored red a little and dyed can beconfirmed F Bacterial plaque is Bacterial plaque is hardly colored red alittle and dyed can be confirmed

1. A composition comprising a peptide consisting of the amino acidsequence of SEQ ID NO:
 1. 2. The composition according to claim 1,wherein the concentration of said peptide is 0.001–20% by weight basedupon the total weight of the composition.
 3. The composition accordingto claim 1, wherein said composition is in a form suitable for oraladministration.
 4. The composition according to claim 3, wherein saidform suitable for oral administration is selected from the groupconsisting of a tooth powder, a toothpaste, a liquid dentifrice,mouthwash, a chewing gum, a gargle, a troche, a cream, an ointment, apasting agent and a tablet.
 5. The composition according to claim 4,wherein said form suitable for oral administration is a toothpaste andsaid toothpaste further comprises one or more additives selected fromthe group consisting of abrasives, caking additives, wetting agents,sweeteners, perfumes, antiseptics, and other pharmaceutically effectiveagents.
 6. The composition according to claim 4, wherein said formsuitable for oral administration is a toothpaste and said toothpastefurther comprises one or more base materials selected from the groupconsisting of water, glycerin, ethylene glycol, diethylene glycol,polyethylene glycol, propylene glycol, polypropylene glycol, sorbitol,xylite, lactite, mannitol, ethanol, and sodium lactate.
 7. Thecomposition according to claim 1, further comprising one or moreabrasives selected from the group consisting of calcium carbonate,calcium silicate, a silica fine powder, amorphous water-containingsilica, hydrophobic silica, calcium secondary phosphate, calciumpyrophosphate, insoluble sodium metaphosphate, aluminum hydroxide, andsilicic acid anhydride.
 8. The composition according to claim 1, furthercomprising one or more surfactants selected from the group consisting ofan anionic surfactant, a nonionic surfactant, an amphoteric surfactant,and a semipolar surfactant.
 9. The composition according to claim 8,wherein said one or more surfactants is at least one anionic surfactantand said anionic surfactant is selected from the group consisting of anα-sulfofatty acid alkyl ester, a water-soluble salt of an α-sulfofattyacid alkyl ester, an alkyl sulfate ester salt, and an N-acylamino acidsalt.
 10. The composition according to claim 8, wherein said one or moresurfactants is at least one nonionic surfactant and said nonionicsurfactant is selected from the group consisting of a fatty acidmonoglyceride, a fatty acid alkanol amide, a polyoxyethylene sorbitanfatty acid ester, a polyoxyethylene hardened castor oil, and asaccharide fatty acid ester.
 11. The composition according to claim 8,wherein said one or more surfactants is at least one amphotericsurfactant and said amphoteric surfactant is selected from the groupconsisting of an alkylbetanine, imidazolinium betaine, and asulfobetaine.
 12. The composition according to claim 8, wherein said oneor more surfactants is at least one semipolar surfactant and saidsemipolar surfactant is an alkylamine oxide.
 13. The compositionaccording to claim 1, further comprising one or more thickeners selectedfrom the group consisting of sodium alginate, propylene glycol alginateester, carboxymethyl cellulose, carboxymethylcellulose sodium, carboxymethylcellulose calcium, starch sodium glycolate, starch phosphate estersodium, sodium polyacrylate, carboxypolymethylene, methylcellulose,crystalline cellulose, hydroxypropylcellulose, polyvinyl pyrrolidone,guar gum, carob bean gum, Tara gum, tamarind seed gum, gum Arabic,tragacanth gum, karaya gum, alginic acid, carrageenan, xanthan gum,gellan gum, curdlan, lactose, chitin, chitosan, and chitosamine.
 14. Thecomposition according to claim 1, further comprising at least onepharmaceutically effective ingredient selected from the group consistingof a germicide, an antiinflammatory agent, a phosphoric acid compoundand an inorganic salt.
 15. The composition according to claim 14,wherein said at least one pharmaceutically effective ingredient is atleast one germicide and said germicide is selected from the groupconsisting of sodium monofluorophosphate, sodium fluoride, chlorhexidinesalts, benzethonium chloride, cetylpyridinium chloride, and benzalkoniumchloride.
 16. The composition according to claim 14, wherein said atleast one pharmaceutically effective ingredient is an antiinflammatoryagent and said antiinflammatory agent is allantoinate.
 17. Thecomposition according to claim 14, wherein said at least onepharmaceutically effective ingredient is at least one phosphoric acidcompound and said phosphoric acid compound is selected from the groupconsisting of sodium phosphate, sodium pyrophosphate, sodiumtripolyphosphate, and sodium tetrapolyphosphate.
 18. The compositionaccording to claim 14, wherein said at least one pharmaceuticallyeffective ingredient is an inorganic salt and said inorganic salt issodium chloride.
 19. The composition according to claim 1, furthercomprising at least one coloring matter or at least one preservative, ormixtures thereof.
 20. The composition according to claim 4, wherein saidform suitable for oral administration is a medicine and said peptide iscontained in said medicine at a quantity ranging from 5 to 50 mg.
 21. Amethod of dental cleaning comprising contacting one or more teeth of asubject in need thereof with a composition according to claim
 1. 22. Amethod of preventing biofilm formation comprising contacting one or moreteeth of a subject in need thereof with a composition according to claim1.